Discipline: Biological Sciences
Subcategory: Physiology and Health
Room: Exhibit Hall
Kassandra R. Ramos - University of Missouri-Columbia
Co-Author(s): Samuel W. Jenkins III, University of Missouri-Columbia, Columbia, MO; Elizabeth A. Grunz-Borgmann, University of Missouri-Columbia, Columbia, MO; Erika M. Boerman, PhD, University of Missouri-Columbia, Columbia, MO
Background: Inflammatory Bowel Disease (IBD) causes chronic immune responses, intra-and extra-intestinal inflammation, and is associated with both increased risk of cardiovascular disease and decreased intestinal blood flow. Perivascular sensory nerves (PSN) dilate mesenteric arteries (MA). Our previous studies indicate that with IBD, PSN function is impaired and may be related to increased immune cells in the MA wall. The aim of this project was to evaluate capsaicin denervation as a means of permanently eliminating PSNs in mice for future studies of immune cell infiltration. Capsaicin, commonly found in chili peppers, targets vanilloid receptor 1 to destroy PSNs that develop in postnatal days 0-3. We hypothesize that capsaicin injections will prevent formation of PSNs in neonatal mice, leading to permanent denervation. Methods: At postnatal days 1 and 2, hypothermia-anesthetized male and female C57BL/6 neonates received subcutaneous capsaicin (50 mg/kg) or sham (control) injections to prevent PSN formation. At postnatal week 6, their MAs were dissected, immunolabeled for calcitonin gene-related peptide (CGRP, sensory nerves) and protein gene-product 9.5 (PGP9.5, all nerves), and then imaged on a Leica TCS SP8 confocal microscope. Fluorescence density for CGRP and PGP9.5 was measured with a custom ImageJ protocol followed by statistical analysis in GraphPad Prism via conventional and nested t-tests. Results: Capsaicin injection was associated with decreased, but not eliminated, CGRP fluorescence vs Sham from 2.4 ± 0.18% to 1.67 ± 0.34% (n=14-18, p=0.056). Post-capsaicin CGRP fluorescence may indicate remaining PSNs, non-neuronal sources of CGRP such as immune cells in the adventitia, or a combination thereof. Nested analysis of CGRP fluorescence showed no significant difference in CGRP between groups, suggesting high within-group variability. As expected, PGP9.5 fluorescence persisted after capsaicin injection (sham: 8.9 ± 0.8, capsaicin: 10.8 ± 1.1%, P=0.188), suggesting that the more abundant perivascular sympathetic nerves were unaffected. Nested analysis showed that capsaicin injection also increased variability within groups for PGP9.5 staining. Conclusion: Capsaicin denervation decreases—but does not eliminate—PSNs on MAs of neonatal mice. Future studies will focus on vascular contractility studies using wire myography and electrical field stimulation to determine whether the observed level of denervation functionally eliminates PSN transmitter release and/or vasodilation.
Funder Acknowledgement(s): This study was supported by The University of Missouri MARC Fellowship T34GM136493 to Kassandra Ramos and R01HL157038 to Erika Boerman.
Faculty Advisor: Erika M. Boerman, PhD, firstname.lastname@example.org
Role: I was involved in every step of the project. I assisted in the administration of the subcutaneous capsaicin injections to the mice, then euthanized them with ketamine-xylazine injections and dissected their mesenteric arteries from the small intestines under a dissection microscope. Then, I performed the immunostaining for confocal imaging and used a confocal Leica Microscope to image fluorescent antibodies. I measured the fluorescence density for CGRP and PGP9.5 with a custom ImageJ protocol and completed statistical analysis in GraphPad Prism via conventional and nested t-tests.